G protein-coupled receptors (GPCRs) are an important family of signal-transducing membrane proteins capable of binding various types of ligands from the extracellular space and activating various signalling pathways inside the cell, rendering them one of the largest protein target families in pharmaceutical research [1]. Receptors of the aminergic GPCRs family are particularly rewarding drug targets as they are implicated in various disease areas, and structure-based drug design has enabled the understanding of ligand binding and function, and the development of more than 500 approved drugs targeting these receptors. Advances in structural biology allowed the determination of more than 300 crystal structures of more than 60 GPCR subtypes to date [2], however, these still represent only a small fraction of known receptor-ligand associations [3].

Site-directed mutagenesis (SDM) is a versatile and frequently employed tool in pharmacological investigations used to infer structural features of protein-ligand interactions [4]. Mutation studies complement structural information provided by crystal structures by defining the roles and relative importance of residues involved in binding, functional activity, and selectivity for ligand chemotypes which have not yet been co-crystallized with their receptors. Community-wide GPCR structure modelling challenges have shown that the best models could be constructed by careful incorporation of mutation and SAR data relating to ligand binding [5]. However, an integrated analysis of receptor and ligand structures and SAR, mutation data, and binding mode prediction has been so far lacking.

The study of Vass et al. can be regarded as a meta-analysis of the site-directed mutagenesis literature for aminergic G protein-coupled receptors [6]. Through an exhaustive database and literature search, the researchers from VU University Amsterdam, Polish Academy of Sciences, University of Copenhagen and Sosei Heptares have collected 6692 mutational data points for 34 aminergic GPCR subtypes of 8 species from 302 publications, covering the chemical space of 540 unique ligands from mutagenesis experiments. This large body of mutation data was also annotated with the structure-based GPCR residue numbering enabling a comparison of mutation effects across different GPCR subtypes and sub-families, and mapped onto the residue positions in the available aminergic crystal structures. Mutation effects were binned into four categories: increased effect, no effect, decreased, and abolished effect, and the data is presented in large overview tables for the five aminergic sub-families. For ligands which had not yet been co-crystallized with their respective receptors, the authors provide predicted binding modes using a combined docking and interaction fingerprint approach to rationalize the mutation effects in light of the ligand SAR.

For each receptor sub-family, a discussion of the known structural receptor-ligand interactions, the ligand chemical space, the structural determinants of receptor-ligand interactions from mutation studies in the amine, major, minor pockets, and the extracellular vestibule, and the possibility of mutation effect extrapolation is provided. The authors also discuss mutation effects of the same ligands across different receptors providing insights into the receptor specific determinants of ligand binding. Finally, an overview is provided of some applications, and the possibilities and limitations of using mutation data to guide the design of novel aminergic receptor ligands.

The authors have deposited the data on Zenodo and in the GPCRdb, and a KNIME workflow was also provided using the 3D-e-Chem KNIME nodes to ease further analysis of the data by the readers [7].

Comments by Chris De Graaf (@Chris_de_Graaf), Director Computation Chemistry, Sosei Heptares.

(1) Santos et al. (2017). A comprehensive map of molecular drug targets. Nat Rev Drug Discov. doi: 10.1038/nrd.2016.230. [PMIDs: 27910877]

(2) Munk et al. (2019). An online resource for GPCR structure determination and analysis. Nat Methods. doi: 10.1038/s41592-018-0302-x. [PMIDs: 30664776]

(3) Vass et al. (2018). Chemical Diversity in the G Protein-Coupled Receptor Superfamily. Trends Pharmacol Sci. doi: 10.1016/j.tips.2018.02.004. [PMIDs: 29576399]

(4) a) Munk et al. (2016). Integrating structural and mutagenesis data to elucidate GPCR ligand binding. Curr Opin Pharmacol. doi: 10.1016/j.coph.2016.07.003. [PMIDs: 27475047] b) Arimont et al. (2017) Structural Analysis of Chemokine Receptor–Ligand Interactions. J Med Chem doi: 10.1021/acs.jmedchem.6b0130. [PMIDs: 28165741]. c) Jespers et al. (2018). Structural Mapping of Adenosine Receptor Mutations: Ligand Binding and Signaling Mechanisms. Trends Pharmacol Sci. doi: 10.1016/j.tips.2017.11.001. [PMIDs: 29203139]

(5) a) Kufareva et al. (2011) Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment. Structure. doi: 10.1016/j.str.2011.05.012. [PMIDs: 21827947]; b) Kufareva et al. (2014). Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment: meeting new challenges. Structure. doi: 10.1016/j.str.2014.06.012. [PMIDs: 25066135]

(6) Vass et al. (2019). Aminergic GPCR-Ligand Interactions: A Chemical and Structural Map of Receptor Mutation Data. J Med Chem. doi: 10.1021/acs.jmedchem.8b00836. [PMIDs: 30351004]

(7) (a) https://3d-e-chem.github.io/; (b) http://doi.org/10.5281/zenodo.58104

Whilst life is always exciting as an ion channel pharmacologist, the last few months have been particularly so, with a large number of publications showing structures of ion channels with regulatory molecules bound to them. In just the last month, the journal, Science, has published several such papers. Three of these concern voltage-gated sodium channels (NaV1.2, NaV1.7) and the binding of potent and selective toxins from animals [1-3]. Another reveals the structure of the primary human cooling and menthol sensor channel TRPM8 bound to synthetic cooling and menthol-like compounds [4].

In the most recent paper [5], Schewe and colleagues extend their outstanding work on selectivity-filter gating of K2P potassium (K) channels (Schewe et al. (2016). Cell. PMID: 26919430), to identify a binding site for negatively charged activators of these channels (styled the “NCA binding site”). Activators which bind to this site open a number of different K2P channels (e.g. K2P2.1 (TREK-1) and K2P10.1 (TREK-2)) and several other potassium channels such as hERG channels (KV11.1) and BKCa channels (KCa1.1), all of which are gated at their selectivity filter. This is exciting, because it is notoriously difficult to design, or even identify, activator compounds for ion channels. This work together with the identification of a separate “cryptic binding site” for K2P channel activators (PMID: 28693035) opens possibilities for rationale design of activator compounds targeting these binding sites, which would provide potential novel therapeutic approaches for the treatment of several conditions including chronic pain, arrhythmias, epilepsy and migraine (PMID: 30573346).

One potential problem, identified by Schewe et al, is the promiscuity of the NCA binding site across several K channel families. However, there are enough structural differences in the region around the NCA-binding site between the channel types to overcome this. Provocatively, Schewe et al even suggest that the simultaneous activation of several different K channel types at once may even be advantageous in certain acute conditions such as ischemic stroke and status epilepticus.

Comments by Alistair Mathie (@AlistairMathie) and Emma L. Veale (@Ve11Emma), The Medway School of Pharmacy

(1) Clairfeuille T et al. (2019). Structural basis of α-scorpion toxin action on Nav channels. Science, pii: eaav8573. doi: 10.1126/science.aav8573. [PMIDs:30733386].

(2) Shen H et al. (2019). Structures of human Na_v1.7 channel in complex with auxiliary subunits and animal toxins. Science, pii: eaaw2493. doi: 10.1126/science.aaw2493. [Epub ahead of print]. [PMIDs:30765606].

(3) Pan X et al. (2019). Molecular basis for pore blockade of human NaNa⁺ channel Na_v1.2 by the μ-conotoxin KIIIA. Science, pii: eaaw2999. doi: 10.1126/science.aaw2999. [Epub ahead of print]. [PMIDs:30765605].

(4) Yin Y et al. (2019). Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel. Science, pii: eaav9334. doi: 10.1126/science.aav9334. [Epub ahead of print]. [PMIDs:30733385].

(5) Schewe M et al. (2019). A pharmacological master key mechanism that unlocks the selectivity filter gate in K⁺ channels. Science, 363(6429):875-880. doi: 10.1126/science.aav0569.. [PMIDs:30792303].

New machine learning algorithm for drug discovery that is twice as efficient as the industry standard and identified potential ligands for the M1 receptor, a potential target for the treatment of Alzheimer’s disease.

A paper from Lee et al. [1] (University of Cambridge) in collaboration with Pfizer, describes the development of an algorithm to use machine learning to separate pharmacologically relevant chemical patterns from irrelevant ones. The algorithm compared active versus inactive molecules at the muscarinic acetylcholine receptor, M1 and uses machine learning to identify components of the compounds are important for binding and which arose by chance. Lee et al. built a model using historic data using ~5,000 compounds that were screened for agonist activity, of which 222 were active. Six million molecules from the e−Molecules database were computationally screened. From these ~100 molecules were purchased and screened in CHO cells expressing the M1 receptor with four compounds identified as agonists (EC₅₀ range 80-300nM).

Comments by Anthony Davenport, IUPHAR/BPS Guide to PHARMACOLOGY, University of Cambridge

(1) Lee AA et al. (2019). Ligand biological activity predicted by cleaning positive and negative chemical correlations. PNAS, https://doi.org/10.1073/pnas.1810847116. [Epub ahead of print]. [PNAS: Article]

G protein-coupled receptors (GPCRs) transduce physiological and sensory stimuli into appropriate cellular responses and mediate the actions of one-third of drugs. Structures of GPCRs are therefore extremely valuable for understanding basic receptor function and rational drug design. Today, 310 structures of 59 distinct receptors (https://gpcrdb.org/structure/statistics) have revealed the general bases of receptor activation, signalling, drug action and allosteric modulation. However, there are still no structures for the vast majority – 85% – of the 398 non-olfactory GPCRs and for 52% structure models can only be based on low-homology templates.

To accelerate the determination of GPCR structures and to help assess the quality of the available templates based on the modifications and methods, a recent article in Nature Methods presents “An Online Resource for GPCR Structure Determination and Analysis” [1]. This surveyes the construct engineering and experimental methods and reagens used to produce all available GPCR crystal and cryo-EM structures. Furthermore, it describes and interactive resource integrated in GPCRdb (www.gpcrdb.org) to assist users in designing new constructs and browsing appropriate experimental conditions for structure studies.

Comments by David E. Gloriam, University of Copenhagen (@David_Gloriam)

(1) Munk C et al. (2019). An online resource for GPCR structure determination and analysis. J Med Chem, doi:10.1038/s41592-018-0302-x. [PMID:30664776]

Cannabinoid receptors respond to multiple endogenous fatty acid derivatives and are often divided into neuronal-associated CB1 receptors and immune cell-associated CB2 receptors. Both receptors are GPCR, coupled predominantly to Gi, and have cytoprotective properties. The predominant psychotropic agent in Cannabis, THC, acts as a partial agonist at both receptors. CB1 patho/physiological responses are often characterised as analgesic, rewarding, orexigenic, hypothermic and amnestic, while CB2 receptors are mostly associated with anti-inflammatory effects.

In many countries, synthetic cannabinoids have become a social issue, with a prevalence of use amongst the homeless and incarcerated, with even a number of deaths attributed to these agents. Although all the molecular mechanisms of action of these synthetic cannabinoids are yet to be defined, one feature they have in common is a high potency and high efficacy profile at CB1 receptors. Kumar and colleagues [1] report a CB1 receptor:Gi complex, where the receptor is bound to a synthetic cannabinoid, MDMB-FUBINACA. The authors report an agonist binding-evoked conformational switch involving residues in TM3 and TM6, which they suggest underlies the high affinity of this synthetic cannabinoid. Furthermore, they conduct in silico simulations to suggest a lateral path of entry for the synthetic cannabinoid between TM1 and TM7 rather than the ‘traditional’ extracellular point of ingress. This lateral diffusion model has been suggested for a number of lipid-binding GPCR.

There is a second cannabinoid receptor crystal structure in the same journal, which focusses on the CB2 receptor [2]. Based on the primary sequences of the two human receptors, there is limited structural identity between CB1 and CB2 (~40 %), although the overlap is much higher in the transmembrane domains, as might be expected, given they bind a number of structurally-diverse ligands with little discrimination (e.g. CP55940, WIN55212-2 and HU210). Li et al report the first crystal structure of the CB2 receptor. In this version, a novel high affinity antagonist/inverse agonist AM10257 was bound to the receptor for crystallisation. The resultant structure shows a number of similarities with the antagonist-bound structure of the CB1 receptor, although notably the extracellular portions of the two receptors diverged markedly. Slightly surprisingly, a close resemblance to the agonist-bound CB1 receptor was identified, which lead them to investigate CB1 receptor function of the novel CB2 antagonist, which turned out to be a low efficacy CB1 receptor agonist.

Comments by Steve Alexander (@mqzspa)

[1] Kumar KK et al. (2018). Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex. Cell, pii: S0092-8674(18)31565-4. doi: 10.1016/j.cell.2018.11.040. [Epub ahead of print]. [PMID:30639101].

[2] Li X et al. (2018). Crystal Structure of the Human Cannabinoid Receptor CB2. Cell, pii: S0092-8674(18)31625-8. doi: 10.1016/j.cell.2018.12.011. [Epub ahead of print]. [PMID:30639103].

Comments by Dr. Nicola J. Smith, National Heart Foundation Future Leader Fellow & Group Leader, Molecular Pharmacology Laboratory, Victor Chang Cardiac Research Institute, Australia

As is often the case with orphan GPCRs, assigning the endogenous ligand has been controversial for the closely related peptide family orphans, GPR37 and GPR37L1. In 2013, Randy Hall and his team (PubMed: 23690594) first reported an association between both centrally-expressed orphan GPCRs and prosaposin (PSAP) and prosaptide (TX14A), the synthetic active epitope of PSAP. Since that time there has been much debate in the field about whether this pairing is correct, with some authors corroborating the findings (PubMed: 24371137; 30010619, 28795439) and others not (PubMed: 23396314; 27072655; 28688853). Note that Head Activator, found in Hydra, was earlier reported as a ligand (PubMed: 16443751) but was quickly discredited (PubMed:28688853; 23686350).

A recent paper by Sergey Kasparov’s laboratory in Bristol has added further fuel to the fire. In a series of well controlled experiments, Liu et al. (PubMed: 30260505) provided convincing evidence that prosaptide is cyto- and neuro-protective and promotes chemotaxis. They are also the first group to demonstrate an effect of prosaptide at a more physiologically plausible potency. At the same time, Bang et al. (PubMed: 30010619) published a ground-breaking paper linking GPR37 expression to macrophage function. Moreover, they proposed a second, more potent ligand for GPR37 (GPR37L1 was not studied): the pro-resolving mediator neuroprotectin D1 (NPD1). Using HEK293 cells expressing GPR37, NPD1 was a potent stimulator of Gαi/o-dependent calcium flux; findings that were corroborated in macrophages isolated from wild type, but not GPR37 knock-out, mice (PubMed: 30010619). Thus, it may be that the endogenous ligand for GPR37 (and perhaps GPR37L1?) is not a peptide after all, but a lipid.

These two studies, while exciting, do little to help us resolve the conundrum that is PSAP/prosaptide and GPR37/GPR37L1. At the very least, it seems likely that prosaptide, if not the highest affinity endogenous ligand at GPR37, is certainly capable of signalling through GPR37 to stimulate Gαi/o signal transduction (whether it is the most potent endogenous agonist will be shown in time as independent groups seek to validate the actions of NPD1).

But what of GPR37L1? This is harder to answer as a number of studies linking GPR37L1 to PSAP/prosaptide have been performed in double GPR37/GPR37L1 knock-out backgrounds or inappropriate tissue models. For example, in the original paper connecting prosaptide to the receptors, the authors claimed prosaptide acted through both GPR37 and GPR37L1 in primary astrocytes, despite the fact that their Western blots demonstrated marked GPR37 expression in comparison to GPR37L1 in the cells (PubMed: 23690594). More recently, they failed to recapitulate this initial pairing in a HEK293 model (PubMed: 28688853). The absence of GPR37L1 in primary astrocytes is consistent with the animal knock-out work of Marazziti et al. (PubMed: 24062445), who showed that GPR37L1 protein was barely detectable before post-natal day 15, which is after the window for isolating primary astrocyte cultures (confirmed by PubMed: 28795439). Coleman et al. (PubMed: 27072655) overcame this expression issue, with difficulty, by using cerebellar slice cultures in vitro to examine Gαs, but not prosaptide, signalling in wild type and knock-out tissue.

In the neuroprotection paper by Liu et al. (PubMed: 30260505) it is clear that prosaptide or PSAP are exhibiting an effect on the cells. By depleting astrocytes of PSAP and then reintroducing prosaptide exogenously, there is an obvious effect on cell migration, cytotoxicity and neuroprotection – phenotypes that are all lost when shRNA knocking down expression of both GPR37 and GPR37L1 are used. Frustratingly, though, the use of a double knock-down approach makes it impossible to ascribe a specific effect to GPR37L1. While GPR37 and GPR37L1 are very closely related by phylogeny and have highly similsar binding sites (PubMed: 27992882), this does not mean that prosaptide is a ligand at both receptors, nor that both receptors signal via the same G proteins (another area of controversy for GPR37L1, where contradictory studies including two by the same team show either Gαi/o or Gαs signaling: PubMed: 23690594, 30260505 vs 27072655, 28688853). Thus, the failure to use single receptor knock-out/knock-downs, or isolate cells with endogenous expression of GPR37L1, represent major limitations in these studies.

Other than the confounding effects of both GPR37 and GPR37L1 deletion in tested cells, what are other reasons that could explain the inconsistencies between studies? Kasparov and colleagues (PubMed: 30260505) attribute this to cellular background, stating that previous studies that failed to confirm prosaptide/GPR37L1 coupling (PubMed: 27072655, 28688853) used HEK293 cells that must be lacking in the necessary endogenous machinery for signal transduction (PubMed: 30260505). To support this claim, they turned to the PRESTO-Tango assay in HEK293 cells to demonstrate prosaptide stimulation could not lead to GPR37L1-dependent recruitment of beta-arrestin. The assay choice is surprising because previous beta-arrestin-based screens at GPR37L1 have failed to show that the receptor can indeed recruit arrestins (PubMed: 23396314, 25895059), and Liu et al. (PubMed: 30260505) did not provide evidence that recruitment was intact in a more physiologically relevant cellular background. Most puzzling though is that the original paper that identified prosaptide and PSAP as GPR37/37L1 ligands used HEK293 cells to make the original pairing (PubMed: 23690594). They also refute the physiological relevance of high constitutive Gαs signalling reported by Coleman et al., even though Coleman et al. demonstrated higher cAMP accumulation in cerebellar brain slices from wild type mice when compared to GPR37L1-/- (PubMed: 27072655).

Further muddying the waters, the physiological role of GPR37L1 itself remains enigmatic. For example, Min et al. (PubMed: 20100464) initially reported GPR37L1 null mice to have a staggering 62 mmHg increase in systolic blood pressure when compared to a cardiac-specific overexpressing model, with the presence of concomitant left ventricular hypertrophy. However, Coleman et al. (PubMed: 29625592) found a far more marginal cardiovascular phenotype, with a small increase in blood pressure evident in female mice only. Notably, male GPR37L1 knock-out mice appeared to be more susceptible to cardiovascular stressors, while females were cardioprotected (PubMed: 29625592). In terms of a developmental phenotype, Marazziti et al. (PubMed: 24062445) found that GPR37L1 null mice displayed precocious cerebellar development with enhanced performance in a rotarod test up to 1 year of age. More recently, though, Jolly et al. (PubMed: 28795439) failed to confirm a behavioural difference in their own GPR37L1 knock out mice. The links between GPR37L1 and neurological defects are also confounded by the fact that GPR37 also needs to be deleted in mice for a clear phenotype to be evident. For example, while a single point mutation in GPR37L1 (K349N) in a highly consanguineous family appeared to be causative of fatal progressive myoclonus epilepsy, the mouse phenotype was most pronounced in double GPR37/GPR37L1 knock-out animals (PubMed: 28688853). In vitro studies of the GPR37L1 K349N mutant found no difference between it and the wild type receptor in terms of receptor expression, processing, signalling or ubiquitination (PubMed: 28688853). In the absence of a transgenic K349N mutant mouse, or any confirmed synthetic agonists or antagonists, the authors then assessed seizure susceptibility in knock-out mice of either GPR37L1, GPR37 or both receptors. Interestingly, using the 6Hz-induced seizure model, the GPR37-/- mice appeared to have a more pronounced phenotype than the GPR37L1-/- mice, while double KO mice were extremely susceptible to seizures at all frequencies tested. GPR37 and double KO mice both displayed more spontaneous seizures, although curiously in the flurothyl-induced seizure model only GPR37L1-/- differed from wild type. Thus, conclusive links between GPR37L1 and a specific physiological or pathophysiological state remain to be provided and it seems in general that we are far from understanding the true biology and pharmacology of the receptor.

A new facet of the human brain has been reported [1] involving a first example of somatic gene recombination in neurons, representing a normal neural mechanism whose disruption could underlie the most common (sporadic) forms of Alzheimer’s disease. Mosaic and somatic recombination of Amyloid Precursor Protein (APP) was identified in this well-known Alzheimer’s disease gene, where increased copies and mutations in rare families or Down syndrome are considered causal. Recombination generates thousands of previously unknown gene variants characterized as “genomic complementary DNAs” or “gencDNAs” that could show identical sequences to cDNAs copied from brain-specific spliced RNAs, as well as myriad truncated forms characterized by exonic deletions and “intraexonic junctions” to produce novel sequences that become “retro-inserted” into the genome of single neurons, with neurons showing from 0 to 13 copies. Recombination appeared to require gene transcription, reverse transcriptase activity and DNA strand breaks. Both forms and numbers of APP gencDNAs were altered and increased in sporadic Alzheimer’s disease. Recombination might normally provide a way to alter post-mitotic neuronal genomes to “record” preferred gene variants for later “playback” without a need for gene splicing, towards optimizing or fine-tuning gene expression, representing a form of memory. The involvement of reverse transcriptase activities implicate potential Alzheimer’s disease therapeutics using reverse transcriptase inhibitors, a possibility supported epidemiologically by relatively rare cases of Alzheimer’s disease in aged HIV patients. Recombination could generate new therapeutic targets. Other recombined genes and affected diseases are possible.

Comments by Jerold Chun, Sanford Burnham Prebys Medical Discovery Institute

(1) Lee MH et al. (2018). Somatic APP gene recombination in Alzheimer’s disease and normal neurons. Nature, doi: 10.1038/s41586-018-0718-6. [Epub ahead of print]. [PMID:30464338].

The cellular thermal shift assay (CETSA) was introduced in July of 2013 as a means to investigate drug target engagement inside live cells and tissues (1). The underlying principle of CETSA is simple – it relies on the thermostability of each investigated protein and how this is altered by ligand binding. Experimentally these changes are assessed by applying a transient heat-pulse to the samples. This results in rapid rearrangements of established equilibria such that proteins denature and aggregate unless stabilised by ligand (1,2). The simplicity of CETSA has allowed prompt adoption in the literature but the importance of rapid changes in ligand binding is still not well recognised.

To explore these considerations we systematically varied both the heat-pulse temperature and duration in CETSA using p38a as our model system (3). Studies spanning seven different heating times and over a 13°C temperature interval show apparent potency changes over four orders of magnitude. These studies demonstrate how quantitative comparisons with functional cellular data require an understanding of the temperature dependence of the interactions under study. Our publications also discuss critical technology developments that allow shorter heating times.  These can now be down in the 10s of seconds range to minimize ligand rearrangements and heat-induced changes to cell permeability.

Comments by Dr. Thomas Lundbäck,  Associate Director, Mechanistic Biology & Profiling, Discovery Sciences, AstraZeneca R&D, Gothenburg,  Sweden,  thomas.lundback@astrazeneca.com. 

1. Martinez Molina, D.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E. A.; Dan, C.; Sreekumar, L.; Cao, Y.; Nordlund, P., Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 2013, 341 (6141), 84-7 (PMID 23828940).
2.  Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Martinez Molina, D., The cellular thermal shift assay for evaluating drug-target interactions in cells. Nat Protoc 2014, 9 (9), 2100-22 (PMID 25101824).
3.  Seashore-Ludlow, B.; Axelsson, H.; Almqvist, H.; Dahlgren, B.; Jonsson, M.; Lundbäck, T., Quantitative Interpretation of Intracellular Drug Binding and Kinetics Using the Cellular Thermal Shift Assay. Biochemistry Nov 2018 (PMID 30418016).

Piezo channels (Piezo1 and Piezo2) are excitatory ion channels which respond directly to a variety of forms of mechanical stimuli. Two recent papers describe some of the critical roles of Piezo channels in sensory neuron transduction (1, 2). In the first, Murthy et al. (1) demonstrate that Piezo2 mediates both inflammatory and nerve-injury sensitized mechanical pain in mice. In the second, Zeng et al. (2), show that both Piezo1 and Piezo2 are responsible for the baro-receptor reflex that regulates blood pressure and cardiac function.

For researchers interested in studying the roles of Piezo channels in mechano-transduction, useful pharmacological tools, at least for Piezo1, in the form of allosteric activators (3) and an inhibitor (4), have already emerged. Named Yoda1, Jedi1, Jedi2 and Dooku1, they are. May the force………

Comments by Alistair Mathie (@AlistairMathie) and Emma L. Veale (@Ve11Emma), The Medway School of Pharmacy

(1) Murthy SE et al. (2018). The mechanosensitive ion channel Piezo2 mediates sensitivity to mechanical pain in mice. Sci Transl Med. doi: 10.1126/scitranslmed.aat9897. [PMID: 30305457].
(2) Zeng WZ et al. (2018). PIEZOs mediate neuronal sensing of blood pressure and the baroreceptor reflex. Science doi: 10.1126/science.aau6324. [PMID: 30361375].
(3) Wang Y et al. (2018). A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel. Nat Commun. 9(1):1300. doi: 10.1038/s41467-018-03570-9. [PMID: 29610524].
(4) Evans EL et al. (2018). Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation. Br J Pharmacol. 175(10):1744-1759. doi: 10.1111/bph.14188. [PMID: 29498036].

The receptor tyrosine phosphatase (RTP) family is a relatively small group of cell-surface proteins with a simple intracellular enzymatic function in the dephosphorylation of phosphotyrosine proteins. There is less known about the endogenous extracellular ligands which regulate RTP activities in physiological conditions, although some RTPs are activated by cell-surface proteins thought to be expressed on neighbouring cells.

This report [1] from the National Institute on Drug Abuse in the USA focusses on the role of RTP type D on reward associated with cocaine administration. They identify that RTP type D heterozygous knockout mice exhibit lower reward responses to cocaine, and that a novel small molecule that appears to target the enzymatic function of RTP type D is able to reduce cocaine-induced place preference and self-administration in wild type, but not heterozygous knockout mice.

Comments by Steve Alexander (@mqzspa)

(1) Uhl GR et al. (2018). β-Subunit of the voltage-gated Ca²⁺ channel Cav1.2 drives signaling to the nucleus via H-Ras. Proc Natl Acad Sci USA, pii: 201720446. doi: 10.1073/pnas.1720446115. [Epub ahead of print] [PMID: 30348770]

The unfortunate French clinical trial disaster in which the FAAH inhibitor BIA 10-2747 (ligand ID 9001)  left one participant dead and several others with serious neurological adverse events, occurred back in January 2016.  However, the primary publication that describes the properties of this lead compound from its Portuguese originators BIAL has only now appeared in September of 2018 (1).  It is more typical for pharmaceutical medicinal chemistry teams to report their initial in vitro optimisation of a clinical candidate well in advance of Phase 1 (i.e. we might have expected to see this paper circa 2017).  While much has been written about 10-2747 in the last two years, very little peer-reviewed data has appeared (see this slide set and blog post).  In this context, the only BIAL journal paper so far on this compound is a disappointment.  The SAR of the series is only reported as % inhibition determinations rather than the more standardised and usefully comparative IC50s (this means we would actually not curate interaction data from such a paper but we have added the reference to the ligand entry). In addition, they do not address experimentally the irreversibility topic over which there has been some confusion. We would also quibble with the use of  “potent” in the title since an independent report of the initial IC50 binding  (i.e. without pre-incubation for inactivation) was only 7.5 uM (2).

Comments by Chris Southan (@cdsouthan)

1. Kiss et al  (2018). Discovery of a Potent, Long-Acting, and CNS-Active Inhibitor (BIA 10-2474) of Fatty Acid Amide Hydrolase.  ChemMedChem, [Epub ahead of print]. [PMID:30113139].
2. van Esbroeck et al. (2017) Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474 Science, 356(6342):1084-1087 (PMID: 28596366)

In this multi-author, multi-centre publication [1] lead by Denise Wootten and Patrick Sexton from the Monash Institute of Pharmacological Sciences, there is reported a 3.3 Angstrom structure of one of the more unusual G protein-coupled receptors. The CGRP receptor is a target for the recently FDA-approved monoclonal antibody erenumab targetting migraine. The receptor is unusual because of its modulation by a trio of accessory proteins exemplified here by RAMP1, which influence both the pharmacological and signalling profiles of the GPCR. Recent structural approaches to studies of GPCR have moved into investigations where a G protein is included. The Liang paper has a Gs heterotrimer attached, and, in addition, has the natural ligand, CGRP bound to the receptor. The report on this six molecule complex thus allows a major advance into the interaction between a GPCR, its endogenous agonist, the requisite G protein AND a modulatory protein.

Comments by Steve Alexander (@mqzspa)

(1) Liang YL et al. (2018). Cryo-EM structure of the active, G_s-protein complexed, human CGRP receptor. Nature, doi: 10.1038/s41586-018-0535-y. [Epub ahead of print]. [PMID: 30175587]

This paper [1] extends previous studies demonstrating a key role of voltage-gated L-type Ca²⁺ channels in the modulation of activity-dependent gene transcription. Earlier work in cultured neurons had already shown that L-type channel activity is required to activate gene expression through different signaling pathways, including the Ras/MAPK pathway [2-4]. This is confirmed in the present study (primarily in experiments with HEK-293 cells) but there are important differences in the way nuclear signaling gets activated. In contrast to previous models [2], it is shown that the Ras/MAPK pathway is turned on by a direct physical interaction of the L-type channel (only Cav1.2 was studied) with Ras through the channel’s beta-subunit. Moreover, this interaction requires binding of extracellular Ca²⁺ to the pore-forming alpha1-subunit but no Ca²⁺ influx through the channel. Also no binding of calmodulin to the channel is required. This strongly suggests a direct conformational coupling of Cav1.2 to Ras activation and adds to other evidence of conformational coupling of L-type Ca²⁺ channels [5-6]. However, further experiments are required to demonstrate that this mechanism also exists in neurons.

Comments by Jörg Striessnig, University of Innsbruck

(1) Servili E et al. (2018). β-Subunit of the voltage-gated Ca²⁺ channel Cav1.2 drives signaling to the nucleus via H-Ras. Proc Natl Acad Sci USA, 115(37):E8624-E8633. doi: 10.1073/pnas.1805380115. [PMID: 30150369]

(2) Dolmetsch RE et al. (2001). Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science, 294(5541):333-9. [PMID: 11598293]

(3) Wu GY et al. (2018). Activity-dependent CREB phosphorylation: convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. Cold Spring Harbor Perspect Biol, 98(5):2808-13. [PMID: 11226322]

(4) Hagenston AM & Bading H (2018). Calcium signaling in synapse-to-nucleus communication. Mol Pharmacol, 3(11):a004564. doi: 10.1101/cshperspect.a004564. [Epub ahead of print]. [PMID: 30021858]

(5) Krey JF et al. (2013). Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons. Nat Neurosci 16:201-9. [PMID: 23313911]

(6) Li B et al. (2016). Sequential ionic and conformational signaling by calcium channels drives neuronal gene expression. Science 351:863-7. [PMID: 26912895]

The potassium channels KV7.2-KV7.5 (KCNQ2-5; GtoPdb target IDs 561-564) regulate neuronal excitability in the mammalian nervous system. The best characterised neuronal KV7 channels give rise to the M current (1) and are mediated predominantly by hetero-tetramers of KV7.2 and KV7.3 subunits (2). Established anticonvulsant agents such as retigabine are known to dampen neuronal excitability by activating neuronal KV7 channels. A tryptophan in the S5 transmembrane region of neuronal KV7 channels is essential for retigabine sensitivity with KV7.3 channels particularly sensitive. Importantly, this residue is not present in the cardiac KV7.1 (KCNQ1) channel, reducing the potential for cardiac side effects.

Now, a pair of studies (3, 4) has shown that this same region of the channel also contributes to a high affinity binding site for GABA and related metabolites (3). GABA activates channels comprised of KV7.3 and KV7.5, including heteromeric KV7.2/7.3 channels, with high potency but low efficacy. GABA does not, however, activate KV7.2 or KV7.4 homomeric channels or cardiac KV7.1 channels. The synthetic anticonvulsant and antinociceptive agent gabapentin activates the same KV7 subunits, although pregabalin does not, despite the overlapping therapeutic efficacy of these two compounds (4).

These results are of considerable pharmacological interest. They suggest a novel mechanism for GABA in regulating neuronal excitability through activation of KV7 channels. They indicate that both GABA and gabapentin will interfere with the action of therapeutically useful activators of KV7 channels such as retigabine. However, perhaps of most value given the relative difficulty in identifying potassium channel openers compared to blockers (5), this work identifies a new chemical class of potential therapeutic activators of neuronal KV7 channels that target an identified region of these channels.

Comments by Emma L. Veale (@Ve11Emma) and Alistair Mathie (@AlistairMathie)

(1) Brown, D.A. & Adams P.R. (1980). Muscarinic suppression of a novel voltage-sensitive K⁺ current in a vertebrate neurone. Nature, 283. 673-676. [PMID: 6965523].

(2) Wang H.S. et al. (1998). KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science, 282. 1890-1893. [PMID: 9836639].

(3) Manville R.W. et al. (2018). Direct neurotransmitter activation of voltage-gated potassium channels. Nature Commun., 9(1). 1847. [PMID: 29748663].

(4) Manville R.W. & Abbott G. (2018). Gabapentin is a potent activator of KCNQ3 and KCNQ5 potassium channels. Mol. Pharmacol., doi: 10.1124/mol.118.112953. [PMID: 30021858].

(5) Liin, S.I. et al. (2018). Biaryl sulfonamide motifs up- or down-regulate ion channel activity by activating voltage sensors. J. Gen. Physiol., doi: 10.1085/jgp.201711942. [PMID: 30002162].

A new type of deorphanization conundrum confronted in pairing the GPCR, MAS1 with the hormonal peptide angiotensin 1–7 (Ang1-7) was emphasised in recent IUPHAR reviews (1, 2). More evidence for disconnection between Ang1-7 and MAS1 is presented in the recent paper by Gaidarov et al. (3). Ang1-7 is produced by ACE2 or neutral endopeptidase by cleavage of a single amino acid, phenylalanine 8, from angiotensin II (AngII), the major renin angiotensin system hormone. MAS1 was described as the primary receptor for Ang1-7 in regulating diverse biological activities, including vasodilatory, cardio-protective, antithrombotic, antidiuretic and antifibrotic effects (4). These activities are lost in tissues of MAS1-deficient animals, producing striking phenotypes observed in the cardiovascular, renovascular, nervous and reproductive systems. Vast physiological responses to Ang1-7 studied in MAS1-deficient animals serve as the most compelling argument in favor of Ang1-7 pairing with MAS1. However, support for a direct interaction of Ang1-7 with MAS1 lack demonstration of classical G protein signaling and desensitization response to Ang1-7, as well as a lack consensus on confirmatory molecular pharmacological analyses (1, 2).

MAS1-selective small molecule agonists and antagonists from Arena Pharmaceuticals have aided mechanistic study of signaling dynamics in recombinant MAS1 expressing cells. Gaidarov et al. systematically tested efficacy of MAS1-selective non-peptide agonists and antagonists in GPCR signaling and also in signaling-pathway independent assay platforms [3]. Arena Pharmaceutical’s non-peptide ligands modulated G protein-dependent and independent pathways through MAS1, including Gq and Gi pathways, 35S-GTPɣS binding, β-arrestin recruitment, Erk1/2 and Akt phosphorylation, arachidonic acid release, and receptor internalization. Moreover, non-peptide agonists produced robust responses in dynamic mass redistribution (DMR) assays that provide a pathway-agnostic cellular response. The Ang1-7 peptide obtained from multiple sources was inert. In the cell-free assay for G protein coupled MAS1, 35S-GTPɣS binding was undetected in the presence of Ang1–7 suggesting lack of direct interaction with MAS1.

To reject the in vivo analysis based MAS1 pairing with Ang1-7 would still be premature based on the conclusions of Gaidarov et al. (and similar papers cited in ref. #2). MAS1 pairing with Ang1-7 should be considered in the context of type 1 and type 2 errors originally described by Neyman and Pearson (5) and recently revisited (6). Type 1 errors are experiments causing rejection of a null hypothesis which may be true. Type 2 errors are experiments insufficient to reject a flawed null hypothesis. Experiments need to be designed to test the validity of MAS1 functions modulated in vivo by Arena Pharmaceutical ligands rather than simply failing to find pairing with Ang1-7. Involvement of additional GPCRs as “Ang1-7 receptors” or signalosome mechanisms that can link Ang1-7 with MAS1 deserve consideration (7).

Comments by Sadashiva S. Karnik (karniks@ccf.org.us) and Kalyan Tirupula

References

1. Karnik SS, Singh KD, Tirupula K, Unal H. (2017) Significance of angiotensin 1-7 coupling with MAS1 receptor and other GPCRs to the renin-angiotensin system: IUPHAR Review 22. Br J Pharmacol. 174: 737-753. doi: 10.1111/bph.13742. Review. PMID: 28194766
2. Karnik SS, Unal H, Kemp JR, Tirupula KC, Eguchi S et al. (2015) International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli [corrected]. Pharmacol Rev 67: 754-819. PMID: 26315714
3. Gaidarov I, Adams J, Frazer J, Anthony T, Chen X et al. (2018) Angiotensin (1-7) does not interact directly with MAS1, but can potently antagonize signaling from the AT1 receptor. Cell Signal 50: 9-24. PMID: 29928987
4. Bader M, Alenina N, Andrade-Navarro MA, Santos RA (2014). MAS and its related G protein-coupled receptors, Mrgprs. Pharmacol Rev. 66: 1080–1105. PMID: 25244929
5. Neyman, J. and Pearson, E.S. (1928). On the use and interpretation of certain test criteria for the purposes of statistical inference. Part I and Part II. Biometrika 20A, 175–240.
6. Lew MJ (2006). Principles: when there should be no difference–how to fail to reject the null hypothesis. Trends Pharmacol Sci. 27:274-278. PMID: 16595154
7. Tirupula KC, Zhang D, Osbourne A, Chatterjee A, Desnoyer R, Willard B et al. (2015). MAS C-terminal tail interacting proteins identified by mass spectrometry-based proteomic approach. PLoS One 10: e0140872. PMID: 26484771

What will Systems Biology look like in the future? Up to now, it has focussed on the development of standards, software tools and databases that enabled us to study the dynamics of physiological function mechanistically. However as these tools and technologies have matured, the focus of the systems biology research community has moved towards how they can best be interconnected and exploited to develop our understanding of health and disease across whole cells, tissues, organs, organisms. This version 2.0 of systems biology, will build on the existing technologies to create resources that are more intuitive, more accurate, more accessible and are easier to use for anyone engaged with research.

Disease maps describe the interactions and pathways that are perturbed from healthy physiological function in disease pathophysiology. The Disease Maps consortia, spearheaded by the Luxembourg Centre for Systems Biomedicine, the Institut Curie and the European Institute for Systems Biology and Medicine, are developing rich resources to enable us to understand how healthy function is perturbed across different scales. These include from the molecular to the organismal, and that embed individually perturbed pathways in a wider intra- and inter-cellular network so that the systems and systemic impact can be more easily investigated [1].   Each disease topic is the focus of a community of clinical, laboratory and systems biology expertise and the consortia is organised as a community of communities with the following adopted principles:-

• Central integration of in vivo and in vitro disease experts across diseases
• Close integration of pathway mapping and modeling expertise
• Regular sharing of best practice and expertise across diseases

The consortia embrace open access, standard formats, modularity, consistency of quality and best practices in the field. It is anticipated that this work will deliver resources that can support comprehensive programmes of systems medicine by including the following:

• Dedicated trusted reference resources describing disease mechanisms that facilitate advanced data interpretation, hypothesis generation, and hypothesis prioritisation.
• Tools for the study of co- and multi-morbidities, which can deliver refined biomarkers for improved clinical diagnostics.
• Tools for the study of systems pharmacology that suggest drug repositioning and multi-drug intervention strategies.
• Novel insights into disease subclassification supporting the development of next-generation disease ontologies.
• Supporting the design and prototyping of new clinical decision-making strategies.

The Disease Maps consortia thus want to accelerate the development of Systems Biology 2.0 and the roadmap presented in this paper describes how it can be steered towards translational utility.

Comments by Steven Watterson (@systemsbiology), University of Ulster

[1]. Mazein A, Ostaszewski M, Kuperstein I, Watterson S, Le Novère N, Lefaudeux D, De Meulder B, Pellet J, Balaur I, Saqi M, Nogueira MM, He F, Parton A, Lemonnier N, Gawron P, Gebel S, Hainaut P, Ollert M, Dogrusoz U, Barillot E, Zinovyev A, Schneider R, Balling R and Auffray C (2018) Systems medicine disease maps: community-driven comprehensive representation of disease mechanisms, NPG Systems Biology and Applications 4:21. [PMID 29872544]

The A1 adenosine receptor is, for most people, a molecular target they can become conscious of when they block it, which happens frequently. Rapid consumption of higher doses of caffeine, in products like Italian espresso or Turkish coffee, provokes a rapid, transient increase in heart rate and a noticeable increase in limb tremor. As the most widely consumed psychoactive substance, caffeine has these effects through blockade of the A1 adenosine receptor, which is found on cardiomyocytes and the peripheral nerve terminals of the sympathetic nervous system (as well as many other locations), leading to an increase in cardiac contractility and noradrenaline release, respectively.

In this report, a 3.6 Å structure of the receptor complexed with the endogenous agonist, adenosine, in the presence of the heterotrimeric G12 protein has been resolved by cryo-EM. As expected, there are differences in conformation compared to the previously-reported antagonist-bound receptor, principally in TM1 and TM2. There are also differences compared to the structure reported for the Gs-coupled, agonist-bound beta2-adrenoceptor.

Comments by Steve Alexander (@mqzspa)

(1) Draper-Joyce C.J. et al. (2018). Structure of the adenosine-bound human adenosine A₁ receptor–G_i complex. Nature, 558. 559–563. [PMID: 29925945]

19 June 2018 update. Announced only about a week after the events described below,  yet a third clinical candidate, lanabecestat  (AZD-3293, LY3314814) has also bitten the dust (4).  The two PhIII trials were stopped because they were deemed unlikely to meet their primary endpoints. This bad news engendered yet another “In The Pipeline” commentary  If detailed reports are eventually published these will be curated as new references for the ligand entry.

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BACE1  (beta secretase 1, BACE-1 or BACE) has been a key target for Alzheimer’s disease (AD) for nearly two decades (1).  However, there was a major disappointment when the Phase III trials with the Merck inhibitor verubecestat failed unequivocally despite lowering A-beta levels.  The termination is reported both in NCT01739348  and the  May 2018  full paper on the trial results (2).  The gravity of this setback is underlined by the “In The Pipeline” commentary title “Merck’s BACE-Inhibitor Alzheimer’s Wipeout” wherein it is suggested that this brings the validation status of this target and, by definition, other inhibitors in late-stage development into doubt.  Thus, even glimmers of success for any mechanistic class of AD  therapy would seem to be currently extinguished.   There remains perhaps the slimmest of hopes from the recent report that the initial process of plaque formation might yet prove sensitive to therapeutic BACE1 inhibition (3).  However, there may be no diagnostic and/or biomarker specific enough to identify prospective asymptomatic patients this early in disease development.

The bad news for BACE1 inhibitors was compounded by a press release from Janssen in the same month. They reported serious liver enzyme elevations for some participants in Janssen’s atabecestat  (JNJ-54861911) Phase 2b/3 trial.  While this may be a chemotype liability for this series rather than a target-related issue, it does mean that yet another AD drug candidate has bitten the dust.  We would hope that a full clinical data report on this trial cessation could be pending,  However,  despite a number of early clinical reports, Janssen has not so far published any primary in vitro medicinal chemistry papers on this compound.  Comments about both these failures have also just  appeared in Nature Reviews in Drug Discovery

N.b. Our BACE1  target entry is in the process of being updated so a number of new inhibitors and curatorial comments will appear in database release 2018.3.  The technicalities of gathering these new structures, including unblinding JNJ-54861911, as well as what might still be progressing,  are described in this blog post.

Comments by Chris Southan (@cdsouthan)

1) Southan and Hancock  (2013) A tale of two drug targets: the evolutionary history of BACE1 and BACE2. Front Genet. Dec 17;4:293. doi: 10.3389/fgene.2013.00293.[PMID 24381583]

2) Egan et. al.  (2018)  Randomized Trial of Verubecestat for Mild-to-Moderate Alzheimer’s Disease. N. Engl. J. Med., 378 (18): 1691-1703, [PMID 29719179]

3) Peters et. al. (2018) BACE1 inhibition more effectively suppresses initiation than progression of β-amyloid pathology, Acta Neuropathol. May;135(5):695-710. doi: 10.1007/s00401-017-1804-9. [PMID 29327084]

4) Update on Phase 3 Clinical Trials of Lanabecestat for Alzheimer’s Disease (2918)  Eli Lilly and AstraZeneca press release [CUL 14602932]

The A2A adenosine receptor is densely expressed in dopamine-rich areas of the brain and in the vasculature. It is the target of an adjunct medication for Parkinson’s Disease, istradefylline in Japan, an A2A receptor antagonist.

The A2A adenosine receptor is an example of a Gs-coupled receptor, activation of which in the cardiovascular system leads to inhibition of platelet aggregation and vasorelaxation. This new report (1) highlights the link between the receptor and the G protein to focus on areas of unexpected flexibility in the ligand binding region. Further, classical understanding of receptor:G protein interaction identifies a prominent role for the third intracellular loop and the proximal end of the C-terminus (in some GPCR, such as the beta2-AR, a fourth intracellular loop is formed by palmitoylation of an intracellular cysteine residue, which the A2A lacks). The model generated from this cryo-EM study with a nanobody suggests a potentially novel role for an interaction between the first intracellular loop and the Gbeta subunit.

Comments by Steve Alexander (@mqzspa)

(1) Garcia-Nafría J et al. (2018). Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein. eLife, 7. pii: e35946. doi: 10.7554/eLife.35946. [PMID: 29726815]

The TRPV2 ion channel is the less well-characterised relative of the TRPV1 or vanilloid receptor that is activated by capsaicin. TRPV2 channels have many similarities to the TRPV1 channels, in that they are homotetrameric and respond to some of the same ligands (natural products such as cannabinoids) as well as being triggered at elevated temperatures. This study (1) focusses on a different common feature of the whole Transient Receptor Potential family, which are often described as non-selective cation channels. Using comparative analysis of crystals structures in which calcium is bound with and without an agonist, resiniferatoxin, present. The authors suggest that this agonist evokes a symmetrical opening of a selectivity filter gate, which permits increased permeation of calcium ions and also larger organic cations, such as the dye Yo-PRO-1.

Comments by Steve Alexander (@mqzspa)

(1) Zubcevic L et al. (2018). Conformational plasticity in the selectivity filter of the TRPV2 ion channel. Nat Struc Mol Biol., 25:405-415. doi:10.1038/s41594-018-0059-z. [Abstract]

Negative allosteric modulators (NAMs) are of great interest in drug development because they offer improved scope for the production of receptor antagonists with enhanced subtype-selectivity. Indeed, many NAMs are already on the market or undergoing clinical trials. NAMs act by binding to sites within receptors that are distinct from the primary, orthosteric ligand binding site and can inhibit the structural rearrangements of a receptor that are induced by orthosteric agonist binding.

P2X receptors are ligand-gated cation channels for which ATP is the endogenous orthosteric agonist. They are expressed throughout the body and the evidence indicates that they have numerous functions, including in sympathetic and parasympathetic neurotransmission, perception of sound, taste and pain, and immune regulation. Seven P2X subunits have been identified, which form trimers, to produce at least twelve different receptor subtypes. A major issue within the field has been a lack of selective antagonists for most P2X subtypes. This is unsurprising given the amino acid sequence similarity within the ATP binding site. Several selective NAMs have now been developed, but little is known about where in receptors they act and how exactly they inhibit receptor activation.

AF-219 is small molecule NAM at P2X3 receptors that was reported to be effective in a phase II clinical trial for treatment of refractory chronic cough. Wang et al., (1) combined X-ray crystallography, molecular modelling, and mutagenesis, to identify the site and mode of action of AF-219. P2X3 receptors are composed of three subunits, each of which adopts a conformation that could be likened to the shape of a leaping dolphin. The tail represents the transmembrane-spanning regions, the upper body the bulk of the extracellular loop and the head the most distal part of the extracellular loop. Also attached to the body are three structurally-distinct elements: the dorsal fin, the right flipper, and the left flipper. As a trimer, the subunits wrap round each other to produce a structure that resembles a chalice.

The AF-219 binding site is formed by the lower body and dorsal fin of one subunit and the lower body and left flipper of an adjacent subunit. Mutational analysis identified which amino acid residues within this pocket are essential for AF-219 binding, whilst in silico modelling showed that the small molecule P2X3 NAMS, AF-353, RO-51, RO-3 and TCP 262, but not the large NAMS suramin and PPADS, also bind to the same site. Activation of P2X3 receptors by ATP closes the binding cavity, so by occupying it, AF-219 prevents the protein structural rearrangements that lead to opening of the P2X3 receptor ion pore.

This identification of the AF-219 NAM binding site in P2X3 receptors is an opportunity for rational, intelligent drug design. It enables virtual screening of compound libraries, with the aim of identifying potential new molecular core structures, which can then be modified in order to optimise the structure of a novel NAM. In addition, this site differs among P2X receptor subtypes, so it is highly possible that drugs with greatly enhanced subtype-selectivity can be developed.

Comments by Dr. Charles Kennedy, University of Strathclyde

(1) Wang J, Wang Y, Cui WW, Huang Y, Yang Y, Liu Y, Zhao WS, Cheng XY, Sun WS, Cao P, Zhu MX, Wang R, Hattori M, Yu Y. (2018). Druggable negative allosteric site of P2X3 receptors. Proc Natl Acad Sci U S A. 2018 pii: 201800907. doi: 10.1073/pnas.1800907115. [PMID:  29674445]